Physical mapping of paar mutations of herpes simplex virus type 1 and type 2 by intertypic marker rescue.

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JOURNALOFVIROLOGY, Aug. 1979, p. 265-276

0022-538X/79/08.0265/12$02.00/0 Vol. 31, No. 2

Physical Mapping of

paar

Mutations of Herpes Simplex Virus

Type

1 and

Type

2 by Intertypic

Marker Rescue

PIERRE CHARTRAND, NIGEL D. STOW, MORAG C. TIMBURY, ANDNEIL M. WILKIE* Medical ResearchCouncilVirology Unit and Department of Virology, University of Glasgow, Glasgow Gll

5JR,

Scotlaud

Received forpublication15January 1979

Mutations (paar) in herpes simplex virus (HSV) which confer resistance to

phosphonoacetic acid involve genes associated with virus-induced DNA

poly-merase activity. Two mutantsof HSV (HSV-1 tsH and HSV-2 ts6) produce a

thermolabile DNA polymerase activity. In this study, the tslesions presentin these mutants and those present in two independent phosphonoacetic acid-resistantmutantsof HSV-1 andHSV-2 (paar_1 andpaar-2) have been physically mapped byrestriction endonucleaseanalysis of recombinants produced between HSV-1 and HSV-2 by intertypic markerrescue.Allfourmutations mapped within

a 3.3-kilobase pair region around map unit40. The accuracy of the method is

reflected by themapping results for tsH andpaar-2, whichwerefoundtoliein thesame1.3-kilobase pair region.paad-1wasfoundtolietothe right ofts6.

Virus-induced DNA polymerase is thought to have a molecular weight of 150,000, necessitating a gene with a coding capacity of 4.6 kilobase pairs. The four

mutationsmappedinthisstudyall liewithinaregionsmallerthanthis, but the

results donotyetprovethatallfour lesions residein thisor anysinglegene.

Phosphonoacetic acid (PAA) inhibits herpes

simplex virus (HSV) replicationin vivo(17),and Maoetal. (12) showed that thedrugblocksthe activity of virus-induced DNA polymerase in vitro. Mutantsof HSV-1 and HSV-2whichare resistant tothe drug (PAAr) have been isolated in several laboratories (6-8), and studies using these mutantshave indicated a direct

involve-mentofvirus-induced DNApolymerase activity

inthemechanismof inhibition.

Hay andSubak-Sharpe (6) firstshowedthat the DNApolymeraseactivity induced by PAA'

mutants was moreresistant to PAA invitrothan that ofwild-type HSV.Resistancetoincreasing concentrationsof thedrugcanbeacquiredby a

multiple-stepprocess (implying several consec-utivemutations), and the increased level of re-sistance in vivoisreflectedin theresistance of DNApolymerase activity invitro (7). Genetic analysis of these consecutive mutations indi-cated very close linkage on the viral genome. Joffre et al. (8) also reported the inability to segregatethe lesions of threeindependently iso-latedPAArmutants. The mostdirect evidence thatthePAAresistancemutation

(paar)

occurs inthestructuralgene of thevirus-inducedDNA polymerase comesfrom the analysis of HSV-1 tsD9 (8, 20, 21). Thistemperature-sensitive (ts) mutant is resistanttoPAA,although it was not

selectedfor thatphenotype, andrevertantsfor the tsphenotypealsorevert toPAAsensitivity

(PAA5).

TheDNApolymeraseactivityinduced

by HSV-1 tsD9 is thermolabile in vivo (1, 8). Recently, Purifoy et al. (20) have shown that

extensively purifiedDNA

polymerase

activityof

HSV-1 tsD9 is both thermolabile and

PAAT,

whereasthe DNA polymerase activity ofa re-vertant,under the same conditions, is

thermo-stable andPAA8.

Analysisofintertypic recombinants between HSV-1 and HSV-2 has proved to be a precise methodfor the

physical mapping

of HSVgenetic markers, including a paar mutation (16) and

virus-induced polypeptides (10, 14-16, 19, 24,

29). Inparticular,

intertypic

markerrescue has beenhighlydevelopedas a meansof

physically

mappinggenetic loci (24).In this communication we describe the precise physical mapping by intertypic marker rescue of

paar

mutations of HSV-1 and HSV-2 and two ts mutations, tsH

(HSV-1)and ts6(HSV-2).Mutants

carrying

the tsH and ts6 markers have been shownpreviously

toinducethermolabileDNA

polymerase

activi-ties (5; I. K. Crombie, Ph.D.

thesis,

University

ofGlasgow, Glasgow,Scotland, 1975).

MATERIALS AND METHODS

Cells andviruses.Babyhamsterkidney (BHK-21

265

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266 CHARTRAND ET AL.

C13) cellsweregrown inEaglemediumsupplemented

with 10% tryptosephosphate broth and 10% calf serum (11).Cellmonolayers (2 x 106 to 4 x 106cells) in 50-mm-diameter plastic petri dishes(Flow Laboratories, Inc.) were usedthroughout.

The ts mutants of HSV-1 strain 17 (tsH) and of HSV-2 strainHG52 (ts6) have been described

previ-ously (13, 25, 26). The HSV-1 strain 17 (ts+ paar-1) and HSV-2 strain HG52 (ts+ paar-2) mutants were those isolated and described by Hay and

Subak-Sharpe (6).

Virus DNA preparation and production of HSV DNA fragments. Virion DNA was prepared fromcellsinfected at310Catamultiplicity of infection of102 as describedbyWilkie(27).

Thepreparationof restriction endonucleasesXbaI,

HindIII,EcoRI,BglII, andHpaIand the conditions used for thecleavageofHSV-1 and HSV-2 DNA were asdescribedpreviously (2, 28).

Intertypic marker rescue and analysis of

re-combinants. We havepreviouslydescribed in detail the technique for intertypic marker rescue (24). Briefly, BHK cellsarecoinfectedatthenonpermissive temperaturewith0.4

jig

of intact DNA of a ts mutant of one serotype and0.4jigof unseparated restriction endonucleasefragmentsof DNAfrom ts+ virus of the other serotype.DNA infectionsarecarriedoutbythe Stow and Wilkie (23) modification of the technique of Graham and Van der Eb (4). ts+ virus progeny from such crosses areplaque purifiedthree times at 38.5°C, and the parental origin of restriction endonuclease sites inthe DNA is determined (19, 24). The rationale behind theanalysisof such recombinants is that DNA sequences from the ts+ parent carrying a ts+ gene sequenceshouldreplacethe DNA sequences ofthets parentatthe site of the ts lesion. In the present study, the DNAfrom thets+ parents carried the nonselected marker(paar)for resistance to 100jigof PAA perml. ts+ recombinantsweretestedfor the presence of this markerby determiningtheirrelativeefficiencyof

pla-quing(EOP)in the presence andabsence of thedrug at38.5and31°C.

Therecombinants described in this studywereall derived fromindependent crosses and were therefore knowntobeclonallyunrelated. Recombinants R6-17, R6-18, and R6-19, which wereisolated from three

well-separatedplaquesfrom the same dish and could have been related, nevertheless proved to have different genomestructures (seeFig.7).

Physicalmaps of HSV-1 and HSV-2. Figure 1 shows the physical maps ofHSV-1 (strain 17) and HSV-2 (strain HG52) for the restriction endonucleases

XbaI,HindIII, EcoRI,BglII,HpaI, KpnI, andBamHI

in theregion 30 to 50 map units of the genome (24; A. Davison,personal communication). One map unit is equivalentto 106daltons (1.7 kilobase pairs) and the genome orientation isasdescribedpreviously (24, 29). Each map indicates theposition of the restriction sites and corresponding fragments, above for HSV-1 and belowfor HSV-2. At the top of Fig. 1 is a summary of all the restrictionsites combined from the seven maps. The numberscorrespond to the numbers given to each restrictionendonuclease, and the sites are aligned with thecorrespondingonesfrom each map. The order of these sites for eachserotype was deduced by

recleav-30

1-XbaI

2-Hind III

3-Eco RI

4-Bgl II

5-HpaI

6-KpnI

7-Bam HI

35 40 45 50

4 5 5 4 15

3

331

3

6 6 6 6 6 6

i7,l7177 717 777 7

7 77 7 77

4 5 5 4 2 4 1

e

c d

a

h e

0 C

t b h

III

j d

a h d

m t n p v a'x c

I

q m t 9 d

p u de g v r kw ib d

In I I

nn'I' b rh'j a yI j

FIG. 1. Physical maps of HSV-1 (strain 17) and HSV-2(strain HG52) for seven restriction endonucle-ases(see text).

ageofindividualfragments (3,28; A.Davison,personal communication). Thealignmentof HSV-1 restriction sites with respect to HSV-2 restriction sites is the result of DNA/DNA hybridization experiments (N. M.Wilkieand R.Cortini,unpublished data)andfrom thestudyof the genomestructureof therecombinants

presentedinthis paper. Thisalignmentcannot,

there-fore, be considered to be completely accurate, al-though thealignedmapsshown inFig.1areconsistent with all of the recombinant structures presented

herein.

RESULTS

Phenotypic characterization of inter-typic recombinants. In markerrescue

experi-ments in which PAA is usedas a selection for recombinants,wehavefoundahighbackground

ofPAAr virus,whichmakesmappingvery diffi-cult (N. D. Stow, Ph.D. thesis, University of Glasgow, Glasgow,Scotland, 1978).Forthis

rea-son, thepaarmutationsweremappedas

nonse-lected markers in recombinants produced by intertypiccrossesbetween intact tsPAA8virus DNA andunseparated restriction fragmentsof ts' PAAr virus DNA, using ability to grow at

38.50Castheselectivepressure.

Tables 1and 2list the 40recombinants ana-J.VIROL.

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MAPPING OFpaar MUTATIONS OF HSV-1 AND HSV-2 267

lyzed

inthis

study,

give

the restriction

endonu-cleases used to

produce fragments

of the ts+

DNA,

and show therelative EOP

38.5°C/31°C

and the relative EOP in the presence and

ab-sence ofPAA for each recombinant and their

parents.

Therelative EOP

38.5°C/31°C

values

ranged

from 0.4 to

5.0,

indicating

that every recombinantwas

ts+.

The relative EOP±

drug

values for

PAAN

recombinants

ranged

from 0.2

to

1.7,

whereas

PAAN

recombinants gave values from 10-4 to

10-3.

The relative EOP ±

drug

determinations shown in Tables 1 and 2 were

carried out at

310C,

but similar values were

obtainedat

38.5°C.

Restrictionendonuclease

mapping

of

in-tertypic

recombinants.

The method of

deter-mining

the

parental origin

of DNA sequencesin

intertypic

recombinants

by

restriction endonu-clease

analysis

has been described

previously

(19, 24, 29).

Three factors must be considered wnen

mterpreung

t.

of DNAfromther(

detected

provided

TABLE 1. Intertyj

ts+ DNA Recombi- fragments

nant produced by: R6-3 HindIII R6-4 HindIII R6-5 HindlIl R6-6 HindIII R6-7 HindlIl R6-8 XbaI R6-9 XbaI R6-10 XbaI R6-11 XbaI

R6-13 HpaI R6-14 HpaI R6-15 HpaI R6-17 EcoRI

R6-18 EcoRI R6-19 EcoRI R6-20 EcoRI R6-21 EcoRI

R6-22 EcoRI R6-23 EcoRI

R6-24 EcoRI

R6-26 BglII R6-27 BglII

R6-28 BglII R6-29 BglII R6-30 BglII R6-31 BgIIl R6-32 BglIl R6-33 BglII

R6-34 BglII R6-35 BglII

HSV-2

(ts6 paa') HSV-1

(ts+ paa-1)

'DNA Cross:HSV-1 paa');selection:ts+ (abi

hEOPin thepresencE

31°C;similarresults at3

TABLE 2. Intertypic recombinants: RHseries'

tsR

DNA Titerat Relative Relative Recombi- fragments 310C EOPEP±

nant produced (PFU/ml) 381.5C/ drug'

by: 10

RH-11 HindIII 3.2x10# 5.0 0.4

RH-12 HindIII 2.5 x108 1.8 0.5

RH-13 HindIII 2.7x108 3.3 0.4 RH-14 HindIII 4.2 x1i0 2.4 0.8

RH-21 HpaI 5.2x108 2.5 0.2 RH-22 HpaI 1.2x108 3.9 0.2 RH-23 HpaI 4.9 x107 2.2 0.4 RH-24 HpaI 9.6 x107 2.5 0.2

RH-31 EcoRI 1.6x107 3.8 0.3 RH-32 EcoRI 9.7x106 3.3 0.3 HSV-1 1.6x107 1.3x10-4 <lo-4

(tsH paa')

HSV-2 2.0xi05 5 0.5

(ts+ paa'-2)

'DNA cross: HSV-1 (tsH paa')x HSV-2 fragments (ts' paa'-2); selection:ts+ (ability to growat38.5°C).

bEOP in the presence (100ug/ml) and absence of PAA at 31°C; similar resultsat38.5°C.

ne

resuits.

rirst,

mepresence clease profile of the putative recombinant ge-escuing fragment willonly be nome is altered from that of the parental ge-that the restriction endonu- nome. Second, definition of the length and po-sition of the inserted rescuingsequences depends

picrecombinants: R6seriesa onthe

alignment

of the

physical

maps for HSV-Titerat Relative Relative 1 and HSV-2 DNA (see above). Third, in cases 31°C EOP EOP± in which the size of the recombinant genome is (PFU/ml)

38.5oc/

drug' unaltered andonly loss ofrestriction endonucle-1.0X i09 0.5 0. ase sites can be found, weassume replacement 1.9x109 0.9 0.5 ofgenomicsequencesof the mutantby rescuing 1.3 x 109 1.2 1.6 DNA sequences at thosesites.

54

xo108

1.2

1.7 Figures 2,

3, 4, and 5 show the restriction 9.9 xlo8 1.3 1.3 endonucleasefragmentpatternsof recombinants

5.7x10 0.8 1.1 in the RH and R6 series. The RH series was

1.9x109 0.8 0.8

5.9x108 1.1 1.0 isolated after the rescue of HSV-1 tsH DNA 6.6x108 1.0 1.7 withDNA fragments of HSV-2 (ts+

paar-2),

and 1.4x109 0.6 1.1 the R6 series was isolated after the rescue of

1.1 x109 1.0 <10-4 2 ts6 DNA with DNA fragments of

HSV-2.6 xio8 0.5 0.7+

7.7 x

108

0.5 0.5 1

(ts'

paar_1).

In the

interest

of

brevity,

only 1.1 x 109 0.9 0.5 those key recombinantswhich define the limits

813

x10"

117

0.24

x10;l of

mapping

areshown. The

analysis

is

presented

6.3x108 1.3 0.7x10-' bydiscussingthe restrictionenzyme mapsof the 9.6x108 0.4 0.3 R6 and RH groups enzyme by enzyme, to show

1.2x

10"

0.8 0.2x

10-4

that the same limited region of the genome is 1.0x109 1.3 1.3 affected in each case. Changes in restriction

7.6x108 0.6 0.9

3.8x108 1.2 1.1

°x

10-l

enzymesitesoutside this

region

willnotbe dis-6.4x108 0.8 1.1 cussed indetail.To aid theanalysis,opencircles

1.2x

i8

0.5 0.5 indicate the loss of fragments compared with the

6.6x

108

1.7 0.6 ts parents, and solid squares indicate the ap-1.4x109 0.6 0.7 pearance of new fragments not present in the ts

2.8x108

<10-_5

1.1X

l0-4

parents. Restriction sites will be

designated by

the two letters that identify the fragment on 8.6x

108

1.1 0.8 either side

(Fig.

1).

From the data shown in

Fig.

2, it can be deduced thatR6-9, R6-14,and R6-30 havelost fragments

(tspaa'-1)

xHSV-2(ts6 the HSV-2 Hindlll h-e site. This produces a

ilit(y100

g/l)andabsenceofPAA at fusion fragment which migrates slightlyslower

38.5°C. thanHSV-2HindlIl a.R6-26showsno change

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HSV1 R6-26 HSV-2

R6 4 R614 R6-30

RH-1i HSV-2

HSV1 H-12

_ _

s

._i

j~~~~~-

._ . ,

.ik:_

J,~~~~~~~~~~~~~~~~~~~~~~

#j: _ _Cdt

...

.}~~~~O

w tJ_.==~~~~1=

.11i

0

---C

Hind III Hirid III

RH 14 HSV-2

HS;V PH 22

*_ C~~ad

cdefg 3 ". I-1

.4.

-U1

b-.a

cde....f

hi<_m

LCU R

FIG. 2. Autoradiographs of32P-labeledDNAofintertypic recombinants and their parents digested with HindIII andEcoRI. TheEcoRIgelisacompositeofdifferentexposuresofthesamegel.Theparentalpatterns

arefrom infections with the actual parents usedtogenerate therecombinants, i.e., HSV-1 (ts+paa'-1)and HSV-2 (ts6paa')forthe R6 series andHSV-2(ts+ paar-2) and HSV-1 (tsHpaa')forthe RHseries. DNA fragmentsareletteredaspreviously (24). Designations:U,newfragments found in recombinants whichare notpresentin thetsparent;0,fragments foundintsparentswhicharemissinginrecombinants;*,fragments

duetosegregationofmixtures present in theparental stockofHSV-1 tsH.

compared with HSV-2ts6. The stock of HSV-1 tsH used consists ofamixedpopulationinwhich

approximatelyhalf the molecules have lost the HindIIIk-isitetogenerate a fusion band which migrates fasterthanHindIII f (bandsdesignated with an asterisk). Cloned recombinants have segregatedinto oneorthe other oftheseforms, and RH-11 and RH-12 show no other change in their HindIIIpatterncompared with the HSV-1tsHparent. The EcoRIanalysesof RH-14 and RH-22 show the lossof the HSV-1EcoRI m-o site, givingrise tolarger fragments.

Figure3showstheanalysiswithBglII. R6-9, R6-14, and R6-26 share the loss of the HSV-2 BglIIofragment. R6-14hasalsolostthe HSV-2BglII j fragment, but this is not the case for

R6-9 and R6-26. It therefore follows that R6-9 and R6-26 have lost the HSV-2 BglII o-csite. R6-30 has a profile similar to R6-9 (data not shown). RH-li and RH-12 show the reverse situation,having acquiredthe HSV-2BglIIo-c siteandconsequentlyasmaller fragment replac-ing the normal HSV-1 BglII d fragment. Al-though notclearon the gel shown, RH-14 has the sameBglIIrestrictionprofileasRH-11 and RH-12. RH-22 haslost theHSV-1 BglII i

frag-mentand hasacquiredtwo newfragmentswith themobilityof HSV-2BglII jand o.

The KpnIanalysisinFig.4showsthatR6-9, R6-14, and R6-30have allacquireda new

frag-mentwith themobilityofHSV-1KpnIv.These recombinants have also acquired a fragment

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MAPPING OF paa' MUTATIONS OF HSV-1 AND HSV-2 269

HSV-1 R6-26

R6-9 R6-14 HSV-2

RN-" HSV-2

HSv_l RH-12

RHI4 HSV-2

HSV-1 RH-22

_ l--~b a.

o~cde C.

Z f d.

e.

f

- h g

hl

ij

_

k..

1

n 0

o -o p

4-#4 4

aI

a

Bgl 11

Bgl

11

FIG. 3. Autoradiographs of 32P-labeledDNAof intertypic recombinants and their parentsdigested with BglII.Fordetails,seethelegendtoFig.2.

which has themobility of HSV-1 KpnIpinthe case of R6-14 and a slightly lower mobility in

thecasesof R6-9and R6-30. This is because all

three recombinants have acquired the HSV-1 KpnIp-vsite, butonly R6-14 has alsoacquired

the HSV-1 KpnI n-p site. R6-26, RH-11, and

RH-12 show no change compared with theirts

parents. The important feature in RH-14 and

RH-22 is the acquisition of a new fragment

which migrates slower than HSV-1 KpnI abcd duetothe lossoftheHSV-1KpnIx-csite.

Theanalysis withBamHI is shown in Fig.5;

in this case the structure of the recombinant

which carries the nonselected paar_l marker (R6-9) and those which are sensitive to PAA (R6-14, R6-26, and R6-30) canbecompared. All

oftheserecombinants have lost HSV-2 BamHI h', but only R6-9 has also lost the adjoining

f

fragment. The three PAA' recombinants have therefore retained the HSV-2 BamHIh'-f site

but lost the b-h' site. It follows that thepaa-i

mutation must lie to the right of the HSV-2 .*;A...ia_

qXp

i.

u 0

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270 CHARTRAND ET

ALo

abc

cl ef

RH-11 HSV-2 RH-14 HSV-2

HSV-I RH-12 HSV-1 RH-22

Sm

_

-

.~~~~~~

rs

::i'XA 4 :i...I...x

It

FIG. 4. Autoradiographs of 32P-labeledDNAof intertypicrecombinants and their parentsdigested with

KpnI. Shown are composites ofdifferent exposuresofthe samegels. The asterisk indicates incompletely digestedDNA. Fordetails,seethelegendtoFig.2.

BamHI b-h' site andcanbe separated fromthe HSV-1ts+counterpartof thets6mutation.The sameregionisinvolvedinthe RHrecombinants. RH-11andRH-12bothhave lostHSV-1BamHI r (difficultto see since rcomigrates with q in this HSV-1 stock), and a new band which mi-grates slower than HSV-1 BamHI b' has ap-peared due to the acquisition of the HSV-2 BamHI b-h' site.Furthermore,RH-12 contains anew fragmentwhich comigrates with HSV-2 BamHIh', and theinsertion of HSV-2 DNA in RH-12therefore includesthe HSV-2BamHI

h'-f'

site. RH-14 and RH-22 show changes in the same region, but in addition have acquired

longersegments of the HSV-2 genome.

By inspection of therestriction mapsshown in Fig. 1, it can be concluded that all of the changesinrestriction sites foundinthegenomes of R6-9, R6-14, R6-26, R6-30,

RH-li,

RH-12, RH-14, and RH-22 are totally consistent with thegenomestructuresshown inFig.6, 7, and8. The genome structures of the other recombi-nants werededucedinthesameway.

Analysisofintertypicrecombinants. The genome structuresindicatingtheparental origin

of the restriction endonuclease sites for seven

enzymes for 35recombinants areshownin Fig.

6, 7,and8.Figure6showsrecombinants isolated from cells coinfected with intact HSV-2 (ts6

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MAPPING OF paa' MUTATIONS OF HSV-1 AND HSV-2 271

HSV-1 R6-14 R6-26

RH-14 R6-9 HSV-2

up

_

to-4w

w

.

4m

R6-30

RH-11 HSV-2

HSV-1 R H-12 RH-22

_.-,~-w_i

l_

ow.-d ~ ~ s-li

S:i

p-

qr-0

0 0

s -

--

...

-*X nseF *; w

j

IV- __{_}

_

L w

W-

__

_.

r_

_ _

:"

....

._ *

rsW

b- Z i jVPvE

0

0.XO

-.d,s. ^Bl#i

^£ M_ .iffeE;ifa

,3i: m :. ...

r -- -> ss-. s;

t

e.

S--e-

4 -

11

- sie

*X_ _ X .s

'F . w ''

s.,'5s'. ,. .:

.... j.

' , _

_3;.:.

BamHI

FIG. 5. Autoradiographs of32P-labeledDNA of intertypic recombinants and their parents digestedwith BamHI.Shown arecomposites ofdifferent gels. DNA fragmentsidentifiedbyA.Davison (personal commu-nication). Fordetails,seethelegendtoFig.2.

fragments of HSV-1(ts'paar-1)DNA. Allofthe is compared with those of the other recombi-recombinants containHSV-1DNAsequences in nantsshown inFig.6, itcanbe concluded that theregion betweenmapunits38and41.Eleven thepaar_1 lesionmustlietotherightof thets+ of the12recombinantsare

phenotypically PAAr,

(HSV-1) counterpart of ts6 (HSV-2).

indicatingstronglinkage betweenpaar_1and the Inanattempttoobtainfurtherrecombinants ts+(HSV-1)counterpartofts6(HSV-2). Recom- which

split

these

sites,

a second markerrescue

binant R6-14 is PAA8 and, assuming that it is

experiment

was carriedout

using

either EcoRI notsimplyaPAA revertantofthepaar-1 (HSV- orBglII fragmentsofHSV-1 (ts+ paa _1)DNA. 1)mutation,inthis recombinant

paa'-i

and the EcoRI and

BglII

have sites within the

region

of ts+ (HSV-1) counterpart of ts6 (HSV-2) have HSV-1 DNAbetweenmapunits38and41

(en-beensplit.When thegenome structure of R6-14 zymes 3 and 4,

respectively,

in

Fig. 1).

The

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30 35 40 45 50

4 5 5

6 6

7I177

6 6

4 15

?I I 11 I

3

747'777 77

313

4 5 5 4 214 1

67 6

5 5

6

6 3 5

6

6 1. 3 5

67 3 5

76 76 6

m m

~~~~~~-7

67 7

6 6

6 7

67 6

7

7 6

4 7

7 6

76 6

7

6

izzzIzI~~zzzzIzIz

counterpartof thets6 lesionmustmap nearthat site.Four other isolates from thiscross (R6-17,

R6-21, R6-22 and R6-23[Table 1]) were

pheno-typicallyts+andPAASbut containedonly HSV-2 restriction endonuclease sites with the seven enzymesused. Inthis casetheywere probably

ts+ revertants of HSV-2 ts6,or they contained

undetected insertionsofHSV-1 DNA.

Recombinants R6-26, R6-27, R6-28, R6-29,

R6-4 R

R6-30,

R6-31, R6-32, R6-33, R6-34,

and R6-35

wererescued with HSV-1 BglII fragments (Fig.

7). Onlyoneisolate(R6-27,Table1) had neither

R6-6 R detectable HSV-1 restriction sites present nor

evidence of HSV-2 sitesmissing;alloftheothers

R6-7 R contained overlapping HSV-1 DNAsequences.

Seven of the nine recombinantscontained

HSV-R6-5 R 30 35 40 45 50

R6-15 R

R6-4 R

R6-11 R

R6-13 R

R6-3 R

R6-9 R

R6-10R

R6-14 S

FIG. 6. Summaryofthegenomestructureof inde-pendent recombinants isolated from the cross be-tweenintactHSV-2(ts6paa)DNA andunseparated restrictionfragments ofHSV-1

(ts'paa'-1)

DNA. For

eachgenomestructure, the HSV-2 DNA sequences

are shown in whiteand the HSV-1 DNAsequence3 areshown inblack,with theregion of uncertainty of theposition ofthecrossovershownas ahatchedarea.

The numbersindicate thedelimitingrestriction sites (above fortypeIand belowfor type 2).The codefor thesenumbers is thesame as inFig. 1. Rindicates PAA',andS indicatesPAX.

genome structures of recombinants obtained from this experiment areshown in Fig. 7.

Re-combinantsR6-18, R6-19, R6-20, and R6-24were

rescued with HSV-1 EcoRI fragments and are

all PAAr. However, in each case the inserted

HSV-1 DNAsequencesspantheHSV-1 EcoRI

site atapproximately mapunit40. The results indicate a strong selection for recombination eventsinvolving HSV-1 DNAfragmentswhich

retainedthisEcoRIsite ("partials" orannealed

ends) and furthersuggest that thets+ (HSV-1)

45 5

3

116 6

1171 777.

4

6 6

7 7

15

3 3 3 6Il 776. 7717

711717

i

45 5 4 24 1

3

67

31

66

6 7

6 6

6 7

6

4

6

6 6

4

6

6 6

4 6

7 6

27

6

4 3x

7

4 3

7/,-7

R6-18 R

R6-20 R

R6-24 R

R6-19 R

R6-33 R

R6-32 R

R6-29 R

R6-31 R

R6-35 R

R6-28 R

R6-34 R

R6-30 S

R6-26 S

FIG. 7. SeelegendtoFig.6.

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MAPPING OFpaa' MUTATIONS OF HSV-1 AND HSV-2 273 30

15

L5

61,l

35 40 45 50

5

6

71

14 15

|7l7lll7

, -

W77

1L1 I7

7

4 5 5 4 2 4 1

3

5 3

6 7

5 4

6

5 4 6

73

74 7

6 6

2 7 7

271.7

3

[image:9.504.57.247.50.431.2]

mziizMM

FIG. 8. Summnaryof thegenomestructureof inde-pendent recombinants isolated from the cross

be-tweenintactHSV-1(tsHpaa") DNA andunseparated

restrictionfragments ofHSV-2(ts'paar-2)DNA.For details,seethelegendtoFig.6.

1DNAsequenceswhichspannedtheBgl site

near map unit 40,which suggests that the

ts'

(HSV-1) counterpartof the ts6 lesion alsomaps

nearthis site.Tworecombinants(R6-26and

R6-34) and HSV-1 DNAsequences which did not

contain thissite, showing thatthe ts+ (HSV-1) counterpart of the ts6 lesion mustmap to the rightofit.Tworecombinants(R6-26andR6-30) werephenotypically PAA' and,unless reversion

of thepadr- mutation is in eachcasethe expla-nation, theserecombinants haveseparated the

paar-l site and thets+ (HSV-1) counterpart of

the ts6 lesion. The genome structures of these

PAA recombinants compared with the PAAr recombinants showthatpad-1 must lie to the

right,atasimilarpositionobtainedby

compar-ing R6-14 (PAAX) with the otherPAAr

recom-binants.

The reciprocal expenment was then carried 6 6

71

6 7

out using the mutant HSV-1 tsH (HSV-1 tsH

doesnotcomplement HSV-2 ts6; L.

L.

Messer, Ph.D. thesis, University ofGlasgow, Glasgow, Scotland, 1978). Recombinants were isolated from cells coinfected with intact DNA from HSV-1 (tsH

paa5)

and restriction endonuclease

fragments

ofHSV-2 (ts+ paar-2) DNA. The ge-nome structures of these recombinants are shown in Fig. 8. All of the recombinants are PAAr indicating strong linkagebetweenpaar-2

and the

ts'

(HSV-2) counterpart of the tsH lesion. Two recombinants (RH-31 and RH-32) wererescuedwithHSV-2EcoRIfiagments but

nevertheless contained the HSV-2 EcoRI site near mapunit40,suggesting thatthe ts+ (HSV-2)counterpartoftsH maps near this site. When theuncertainty in the locationof the crossover

points istakeninto account,allofthe recombi-nants of the RH series had HSV-2 DNA se-quencesm commonwithin the HSV-2 BamHI h'fragment.

Summary

of

analysis.

In the

analysis

shown

in Fig. 6, 7,and 8, we have mapped the HSV-1 DNA sequences (or HSV-2 DNA sequences) which correct the ts lesion in HSV-2 ts6 (or

HSV-1 tsH). We assume that the correction

mechanismusually involvesthereplacementof

defective sequences in the tsparent by the ts+ counterpartfrom the other serotype. Other

ex-planationsarepossible, but if the assumption is correct,the map location of the ts6 mutation is

defined by the HSV-1 DNA sequences which the R6series ofrecombinantshave in common.

Inspection of the data shown in Fig. 6 and 7

indicatesthat thisregion is defined onthe left

by the HSV-2 HindIII h-e site or the HSV-1

Bgll

i-dsite(R6-26andR6-34) andontheright

by the HSV-2 BamHI h'-j' site (R6-14, R6-26, and R6-30; see Fig. 6, 7, and 1). The region of the genome containing the tsH lesion can be

derived in the same way by inspection of the datafortheRH series(Fig. 8)and is definedby

the HSV-2 BamHI h'fragment,which isa

frag-mentof 1.3kilobasepairs. These data are sum-marized in Fig. 9; note that the limits shown take intoaccounttheuncertaintyinthe location of thecrossoverpoints.

Figure9 also

sumarizes

the

maximum

map

coordinates for the HSV-1 (paa

-1)

and the

HSV-2

(paar-2) paar

mutations. Allof the RH

series ofrecombinants acquired PAArand

pre-sumably

contained thepaar-2 mutation. Thus,

the map coordinates which define thepaar-2 mutation are the same as those for the tsH lesion. Three R6 recombinantswerePAA',and therefore

thepaa-1

mutationwas

split

fromthe ts' (HSV-1) counterpart ofts6. Comparison of the genome structureofR6-14,

R6-26,

and

R6-VOL. 31,1979

3

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30with the remainingrecombinants in the R6 series showsthatpaar_1 mustmapattheright

of ts6 between the coordinates shown inFig.9. These are defined on the left by the HSV-2 BgIII o-c site and onthe right bythe HSV-1

KpnIx-csite(see Fig.6, 7, and1).

DISCUSSION

This communication describes the detailed physical mapping of mutations (tsH and ts6)

present in ts mutants of HSV-1 and HSV-2 which inducethermolabile DNA

polymerase

ac-tivity (5; Crombie, Ph.D. thesis) and those (paar_1 andpaar-2) present intwomutants re-sistant to PAA. Bothphenotypesareconsistent with lesions in genes which affect viral DNA

polymerase activity insome way.The method of mapping involved genetic analysis of

inter-typic recombinants combined with restriction endonuclease mapping of their DNA. The

ra-tionale behindsuchanapproachhasbeen stated

previously (19, 24; thispaper), but it is

worth-40

6

7 6

paat2 tsH

I I

3, 3

6,.

3

6 771

111

II I

17

7 7,

6 6 ' 3,

24,3

Il

ts6

r

paa-I

[image:10.504.96.215.315.607.2]

40

FIG. 9. Summary ofthemappingdatafor tsH,ts6,

paar_1, andpaad-2. Theregion ofthegenomeshown

isanenlargement ofthe 39- to 43-map unitregion shown inFig. 1.

while to consider in more detailwhat is actually being mapped in such experiments. The inter-pretation of mapping results for ts lesions is based on the assumption that rescuing DNA sequences contain the ts+ counterpart of the ts mutation and thattheyreplace sequences at the site ofthe mutation. Thisinterpretationwould be invalid if "second site" rearrangements re-sulting in inter- or intragenic suppression were commonly responsible for the observed pheno-types of the recombinants. The recombinants isolated in this study show a wide range in the length of rescuing DNA sequences, which argues against crossovers whichresult inintragenic sup-pression of the mutantphenotype. Another gen-eral drawbackto the mapping rationalewould be in cases in whichheterozygosity ispossible. Inthis case ats+ phenotype couldbeacquired

withoutloss of the gene coding for the ts phe-notype. In the present work, thereciprocal map-ping of HSV-1 and HSV-2 mutations whichlie

in the same complementation group (tsH and ts6

[Messer,

Ph.D.

thesis])

puts both mutations in aregion of DNA smaller than that thought necessarytocode for theexpectedprotein(DNA

polymerase [see below]). Since intratypic marker rescue experiments using isogenic ma-terial also put both mutations into the same region (P. Chartrand, N. M. Wilkie, and M. C.

Timbury, manuscript in preparation; Stow,

Ph.D. thesis), this reduces the likelihood that

heterozygosityisaproblem forthislocus.

Inter-pretation of the mapping of paar (the nonse-lectedmarker) depends onlyontheassumption that inserted DNA sequences contain the paar mutations.

Given that the assumptions are correct, the

mapping studies with tsH and ts6 delimit the locationfor both thetslesions.Theclones which contain inserted HSV-1 DNA sequencesallow mapping of thepaar_1 (HSV-1) mutation, and those with HSV-2 DNA insertions allow the mapping of the paar-2 (HSV-2)mutation.From

Fig.9itcanbeseenthat all four mutations map in one region of HSV DNA, which spans ap-proximately2 x106daltons (3.3 kilobase pairs). When intratypic marker rescue (isogenic sys-tem) was used, the mapping of tsH and ts6 mutations gave very similar results (Chartrand

et al., manuscript in preparation; Stow, Ph.D. thesis).

This is the first report of the simultaneous and independent mapping of an identifiable function of HSV-1 andHSV-2 (PAA'),and the resultsstrongly suggest closelysimilar map lo-cations. Theuncertaintyinthefinealignmentof the HSV-1 and HSV-2physical maps may ob-viously affect theinterpretation,butit should be

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MAPPING OF

paa'

MUTATIONS OF HSV-1 AND HSV-2 275 noted that themaplocations for the mutations

tsH, ts6, and paar-2 are defined by restriction

endonuclease sites in the HSV-2 physicalmap.

Recent studiesutilizingHSV-1/HSV-2 inter-typicrecombinants havepermitted the mapping of several HSV-induced polypeptides (14, 16).

The order ofpolypeptides in the region under study (14) was found tobe Vmw 117 (glycopro-tein), Vmw 136'(130) (DNA bindingprotein), and Vmw28. In the Marsden etal. (14) study, five

intertypic recombinants isolatedafterrescueof

HSV-1 tsHwereanalyzed. The onlypolypeptide

whichneversegregated fromthe rescuing DNA

sequences wasthe Vmw 136'(130) DNA binding

protein. This probably excludes the genes for

theotherpolypeptides from containing the site forthetsHmutation, butit doesnotprovethat

the gene for the V,w 136'(130) protein does

contain the site for the tsH lesion.Purified viral DNApolymerase has beenreported tohave a

molecularweightof150,000(18), but it hasnot yetbeenassigneda maplocus.

Sincemutantscarrying the tsH,ts6,paa-r1,or

paar-2 mutation show phenotypic properties compatible with lesions in virus-induced DNA polymerase activity (5, 6; Crombie, Ph.D. the-sis), thequestion arises whether thefour lesions

are inthe same gene. tsH andts6do not

com-plement each other (Messer, Ph.D. thesis) and, provided unforeseencomplications donotarise inintertypic complementation tests, these

mu-tationsarelikelytoreside in thesame gene.The paa-r andpaar-2 mutations lie very near the

tsHandts6 lesions. Thecodingcapacity needed for the 150,000-molecular-weight polypeptide whichPowelland Purifoy (18) found ina

puri-fied preparation of HSV DNA polymerase is approximately4.6kilobasepairs.The maximum mapping limits (3.3 kilobase pairs; Fig. 9) are

thereforeconsistentwithallfourmutationslying

inthestructuralgeneofthe viral DNA polym-erase,but donotprovethathypothesis. A paar mutation hasrecently been mapped (9), using intratypicmarkerrescue,andreportedtolieto

theright of the HSV-1HpaI b-h site (map unit 45;seeFig. 1). This isatleast3.8kilobasepairs

tothe right of thepaar lesionsreported in this

study. This raises the possibility thattwo inde-pendentgenescancarrymutations which result

inaPAAF phenotype. If the PAArphenotype is

the result of modifications in the viral DNA

polymerase, this mayimplytwoloci whichare

involved in DNA polymerase activity. In this respectit is interesting to notethattwots

mu-tations(tsC4 andtsD9) which lieintwodifferent HSV-1 complementation groups (22) induce a

viralDNApolymeraseactivity which, when

ex-tensively purified,isthermolabile in vitro (20; K.

L.Powell and D.J. M. Purifoy, personal

com-munication).ThetsD9mutation, which also

re-sultsin a PAA'phenotype(seeabove),has been found to map between 0.30 and0.45 (fractional

length)(D. S. Parris and P.A.Schaffer, personal communication). Although this is consistent with our mapping data, more precise physical mappingand furthercomplementationtestswill berequiredtoresolve theprecise relationshipof the differentpaar mutations and the tsH, ts6,

tsD9,andtsC4lesions.

ACKNOWLEDGMENTS

WethankM.MurphyandC.Adair for cheerful and expert assistance, A. Davison for thepreparationof restriction en-donucleases, and J. Hayforproviding seed stocksof the PAA' mutants.Wealso thankK.L.Powell, D. J.M.Purifoy,D.S. Parris,and P. A. Schaffer for thepersonalcommunication of unpublished data.WearegratefultoJ. H.Subak-Sharpefor his interestthroughoutthisstudy andforvaluablecriticism of themanuscript.

P.C. is therecipientofafellowship from the Canadian MedicalResearchCouncil.

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